Lung cancer is the most common malignancy both in incidence and mortality globally. American Cancer Society's 2017 estimate suggest that there will be 222,500 new diagnoses and 155,870 deaths from lung cancer this year in the United States alone. Unfortunately, lung cancers are often driven by oncogenic alterations that are either not suitable for direct targeting or for which resistance invariably develops to currently approved targeted therapies. Thus, novel strategies for treating lung cancer are urgently needed. The goal of this application is to discover new approaches targeting a subtype of lung cancer with defects in PPP2R2A, a regulatory subunit of the heterotrimeric serine/threonine tumor suppressor phosphatase PP2A that has diverse functions in the regulation of key intracellular signaling processes including the negative regulation of numerous oncogenic signaling pathways such as AKT, MAPK, and MYC. PPP2R2A expression is frequently reduced in a wide range of human cancers. Specifically, 43% of lung cancers display decreased PPP2R2A expression. Most importantly, a defect in PPP2R2A is often associated with poor prognosis. Our recent novel genome-wide synthetic lethality screen identified that PPP2R2A loss of function leads to sensitivity to an inhibitor of CHK1, a key downstream effector of the cell cycle checkpoint protein ATR. Currently, while ATR and CHK1 inhibitors are in clinical trials, there is no specific target based biomarker to identify treatment responsive populations. In this application, we hypothesize that ATR and CHK1 inhibitors specifically target PPP2R2A-defective lung cancer cells by enhancing replication stress resulting in synthetic lethality. The decreased PPP2R2A function could represent a biomarker for the identification of treatment responsive patient populations to ATR and CHK1 inhibition. Oncogene activation can cause replication stress and subsequent double strand breaks (DSBs), the most dangerous type of DNA damage. Thus, cancer cells with increased replication stress heavily rely on ATR/CHK1 for survival as this signaling network is able to modulate replication stress to less toxic levels. Given the role of PPP2R2A in negatively regulating multiple oncogenic pathways, PPP2R2A defects may lead to oncogene activation and replication stress, rendering these cells sensitive to ATR/CHK1 inhibition. To test our hypothesis, two Specific Aims are proposed. Aim 1 is to assess the anti-tumor activities of ATR and CHK1 inhibitors by determining the efficacy of ATR/CHK1 inhibition in suppressing the growth of lung cancer cells with or without a deficiency of PPP2R2A, using clonogenic assays, cell line-based xenografts, and patient-derived xenografts (PDXs). Aim 2 is to evaluate ATR or CHK1 inhibition-induced replication stress in lung cancer cells with or without a deficiency of PPP2R2A, by measuring elongated single strand DNA (ssDNA)/DSBs, replication dynamics and dNTP pools. Combined, if successful, our study could have a significant impact on improving the survival of lung cancer patients, by identifying new approaches to targeting and provide a new stratification biomarker for identifying ATR/CHK1 inhibitor sensitive patient populations in clinical trials.